Process for preparing a streptokinase



United States Patent 3,186,920 PRGCESS FQR PREPARlNG A STREPTOKINASE Norbert Heimhurger, Cappel, near Marhurg (Latin),

Rudolf Schmidtherger, Marhach, near Marhurg (Lahn), and Gerhard Schwich, Marhurg (Icahn), Germany, assignors to Behringwerke Aktiengesellschait, Mmhurg (Lahn), Germany, a corporation of Germany N0 Drawing. Filed Feb. 7, 1963, Ser. No. 256,808 Claims priority, application Germany, Oct. 17, 1962,

5 Claims. (a. 195-.-66)

For preparing a streptokinase of high potency, it is necessary to convert the crude streptohinase into a highly pure streptokinase by appropriate purification measures. However, the purification must be carried out under sterile conditions to ensure that the streptokinase be tolerated intravenously. Hence it is of importance to prevent contamination of the streptokinase by pyrogenic substances, i.e. substances which cause fever. In addition to good tolerance on intravenous injection, the isolated product should also have a high potency.

Now, we have found a process for preparing an immunologically uniform streptokinase under steril conditions, wherein a streptokinase-containing aqueous buffer solution obtained from a streptococci filtrate and having a molarity of 0.01 M and a pH-value of 5.0, preferably a sodium citrate butter, is adsorbed on a carboxymethylpolysaccharide which has been heated twice for 3 hours to 60 C., the interval between these heating periods being two hours, unspecific proteins that are still in solution are eliminated, the main fraction of streptokinase is eluted with a 0.02 molar buffer solution having a pH-value of 5.5, preferably a sodium citrate bufier solution, and the residual fraction of streptokinase is eluted from the carboxymethylpolysaccharide with a 0.1 molar buiier solution having a pH-value of 65, preferably a sodium citrate butter solution.

The process is carried out according to the following scheme:

Initial streptolrinnse solution buffer solution 0.01 M pH-5.0

Adsorption of the streptokinase main traction on carboxyrnethylpolysaeeharide L Elution 0.02 M butler solution pH 5.5

Solution rejected,

contains unspecific proteins and a small amount of streptokinase only Elation 0.1 M buffer solution pE 6.5

The process of the present invention may be carried out in a column, for example, in a pressure filter having the form of a column, or according to the batch process. In general, pressure filters consist of a cylindrical tube the upper third of which is conical and whose base plate is the filter. With such pressure filters the liquids are filtered at an excess pressure to prevent evaporation.

In the process of the present invention, there may be used suitably a commercial filter, for example, one having a capacity of 240 cc., a height of mm., a plate diameter of 60 mm. and a discharge pipe with a diameter of about 11 mm.

As adsorbing agents, there may be used carboxymethyl-polysaccharides, for example, carboxymethylcellulose or cross-linked dextran etherified with carboxymethyl groups.

A special advantage of the process of the invention is that it may be carried out within the short period of 4-5 hours and thus even at room temperature. An immunologically uniform streptokinase is obtained which is suitable for intravenous application and which is free from streptodornase, streptolysin, hyaluronidase, and diphosphopyridinenucleotidase.

It is advantageous to start from a streptokinase which has been purified in known manner, for example, by alcohol and ammonium sulfate precipitation. After application of the process of the present invention, there is then obtained a streptokinase having almost 800 units/ gamma N. The potency is expressed in Christensen units.

The immunological uniformity of the streptoltinase obtained according to the present invention is proved by immuno-electrophoresis andby the Ouchterlony test.

The following examples illustrate the invention, but they are not intended to limit it thereto:

Example 1 A pressure filter having a capacity of 240 cc. was filled up to two thirds of its volume with a carboxymethylcellulose which had been treated twice for 3 hours each at 60 C., with an interval of two hours, and which had been equilibrated, i.e. rinsed with a 0.01 molar citrate buffer having a pH-value of 5.0 until the eluate showed a pH-value and a specific conductivity similar to that of the buffer (1.45 M S/cm.). v

The column prepared in this manner was then charged with 60 cc. of a concentrate of a streptococci filtrate in a 0.01 molar sodium citrate buffer solution containing about 1% of protein, thus 37 million units of streptokinase per'60 cc. of solution and 385 units of streptokinase per gamma N, rinsed with a 0.01'molar citrate butler having a pH-value of 5.0, until spectrometric tests showed the rinsing liquid to contain no more proteins. The rinsing liquid contained the main quantity of unspecific proteins and in addition thereto 2.2 million units of streptokinase (5.9% of the quantity of streptokinase initially used).

The adsorbed streptokinase was then eluted in two stages; first with 650 cc. of a 0.02 molar citrate buffer having a pI-I-value of 5.5, wherefrom 15 million units=40L5% (730 units per gamma N) were obtained, and eventually with 400 cc. of a 0.1 molar citrate buffer having a pH-value of 6.5, whereirom 7.8 million unit-s=21% (590 units per gamma N) were obtained.

Thus, from the total of 67.4% streptokinase recovered,

61.5% were contained in these two streptokinase fractions. 40.5% of the recovered streptokinase showed approximately double the potency of the starting material. lmmuno-electrophoresis and the Ouchterlony test proved the product to be uniform.

Example 2 To 500 mg. of a crude streptokinase which had been purified by alcohol fractionation and which showed a 3 biological activity of 350 units per gamma N, dissolved in a 0.01 molar sodium citrate buffer having a pH-value of 5.0, were added 50 g. of a carboxymethylcellulose which had been equilibrated with the same buffer and heated for 6 hours to 60 C.; the whole was then stirred mechanically for one hour at room temperature. The carboXyrnethyl-cellulose/streptokinase complex was then separated from the solution by centrifugationand Washed .with 250 cc. of the 0.071 molar sodium citrate butterhaye ing a pH-valuerof 5.0. There followed a first elution of the adsorbed streptokinase with 2 200' cc. of a 0.02

:molar sodium citrate butter having apH-value of 5.5

and subsequently a second elution with 2 200 cc. of a 0.1 molar'sodium citrate buffer having a pH-value of 6.5;

The two eluates which contained the d'esorbed streptoa diameter of 1.9 cm., the

lsinase were then separated, from "the carboxyrnethylcellulose by centrifugation. Thestreptokinase content of both solutions was determined; the following values were obtained: 7

7 s Yield 1) 12 'million with 790 units per gammaN 43%. (2) 7.3 million with 630 units .per vgamma'N 26%. L

i 69% of the starting'product.

From the-19.3 million units of streptokinase recovered, 62% proved uniform on immuno-electrophoresis.and in the Ouchterlonytest. The remaining 38% showed in the Ouchterlony test a slight amount of another protein. This component couldvbe eliminated to agreat extent by repeating the'adsorption and elution of the product as de scribed above. 1

Example 3 a 100 cc. of a 0.5% protein'solution obtained by ultrafiltration from a streptococci filtrate, which thus contained 40,000 units per cc., i.e. a total of 40 million units of streptokinase and 50 units per gammaN, were purified on carboxymethylcellulose as described iniE ramples 1 "units '(400 units per gamma N),,';th us 22.5% of the s initial streptolrinase content.

The principal fractionthus contained a product was 12 times as pure as the starting product.

6 cc. of a 1% streptokinase solution purified with am? monium ,sulfate and alcohol and containing 700,000 units per ceand 435units .pergamma N, we r e purified as described in Example 1,.buton a cross-linheddextran which i etherified with .carboxymethyl groups. The adsorbing agent was contained in a glass tube of 20 cm. leng t h and I streptokina-se'. 45

i 1960), Photo-Copy 16.7,Dex.

I Library.)

lower part of thetube, lo. 2 cm., being conical to a diameter of 0.4 cm. I

There were obtained: 7

The first batch containing 0.8 million units (7 units per gamma N), thus 19% of the initial content of non- E adsorbed streptokinase and unspecific proteins;

'The principal fraction as the first eluate of the' strepto-' kinase adsorption product, containing 1.642'I1'll1ll011 units (720 units per gamma N), thus 39% of the. initial streptokinase content;' E The'final batch-asithe second eluate containing 0.93 million ,units (500 units per'gamma N), thus 22% ,ofthe initial streptokinase content. 7 l

Thus, the starting material which-already contained 435 units per gamma N was purified to 1 /2 times ofits biological activity.

We claim: v

1. The process for preparing an immunologically unif rm Streptokiriaseunder sterile conditions, wherein a streptokinase-containing aqueous'bufier solution obtained from astreptococci filtrate and having a molarity of 0,01

and a pH-value of 5.0, is adsorbed ona'carboxymethyl- 'polysaccharide selected fromthe group consisting of carboxyrnethylcellulose and crossilinked dextran etherified with'carboxymethyl groups, which has been heated twice for 3 hours each to 60 C., with an interval of two hours,

unspecific proteins that are still in solution are eliminated,

the principal fraction of streptokinase is eluted from the carboxyrnethyl-polysaccharide with a 0.02 molar buffer solution havingapH-value of 5.5 andthe residual fraction of streptokinase is eluted with a 0.1 molar bufier solutionhaving a pH-value of 6.5.

2. Theprocess as claimed in claim 1, wherein theproc V essis started from 'a' streptokinase purified in knownfrnanner, for example, by; alcohol and ammonium sulfate precipitation.

3. The process as claimed in clairnl, wherein carboxymethylcellulose is us'ed'as carboxymethyl-polysaccharide for adsorbingthestreptokinase.

. .4HIihe process as claimed in claim 1, wherein crosslinked dex'tr'an etherified with carboxymethyl 'groups is used .as carbo xymethyl-polysaccharide for adsorbingthe SLTheiprocess asclaimed in claiml, wherein'an aqueous sodium citrate solution used as bufier solution. I

7 Re erence Cited b t ell al insr UNITED STATES PATENTS P 3,042,586 7/62 Siegel et al. -66 3,042,667 "7/62 Pgedinetal. 260-209 3,107,203 10/63 Baumgarten et al. 195 -66 7 H R RE ERENC Flodin et al.: Nature, 188, 493-494 (NovemberS,

(Available in Patent fice Science Library.)

Porath et al.:"Nature 191,69 7-0 (Iuly 1, 1961), Photo- Copy 167 Dex. (Available in'the Patent Oflice Science A. LOUIS MONACELL, Primary Examiner. NORMAN G. TQRCHIN, Examiner. 

1. THE PROCESS FOR PREPARING AN IMMUNOLOGICALLY UNIFORM STREPTOKINASE UNDER STERILE CONDITIONS, WHEREIN A STREPTOKINASE-CONTAINING AQUEOUS BUFFER SOLUTION OBTAINED FROM A STREPTOCOCCI FILTRATE AND HAVING A MOLARITY OF 0.01 AND A PH-VALUE OF 5.0, IS ADSORBED ON A CARBOXYMETHYLPOLYSACCHARIDE SELECTED FROM THE GROUP CONSISTING OF CARBOXYMETHYLCELLULOSE AND CROSS-LINKED DEXTRAN ETHERIFIED WITH CARBOXYMETHYL GROUPS, WHICH HAS BEEN HEATED TWICE FOR 3 HOURS EACH TO 60*C., WITH AN INTERVAL OF TWO HOURS, UNSPECIFIC PROTEINS THAT ARE STILL IN SOLUTION ARE ELIMINATED, THE PRINCIPAL FRACTION OF STREPTOKINASE IS ELUTED FROM THE CARBOXYMETHYL-POLYSACCHARIDE WITH A 0.02 MOLAR BUFFER SOLUTION HAVING A PH-VALUE OF 5.5 AND THE RESIDUAL FRACTION OF STREPTOKINASE IS ELUTED WITH A 0.1 MOLAR BUFFER SOLUTION HAVING A PH-VALUE OF 6.5. 